Differential expression of hERG1 channel isoforms reproduces properties of native I(Kr) and modulates cardiac action potential characteristics

Abstract

Background: The repolarizing cardiac rapid delayed rectifier current, I(Kr), is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I(Kr). Marked heterogeneity in the kinetic properties of native I(Kr) has been described. We hypothesized that the heterogeneity of native I(Kr) can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD) and restitution.

Methodology/principal findings: The results show that the heterogeneity of native I(Kr) can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased.

Conclusions/significance: Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I(Kr) kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I(Kr). Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.

Figure 1. Topology of the hERG1 Markov model.

The hERG1 Markov model developed by Fink et al. , consists of three closed states (C₁-C₃), an open state (O) and an inactivated state (I). Allowed transitions between states are indicated with arrows. The transition rates are indicated above or below the corresponding transitions.

Figure 2. The relative abundance of hERG1a and hERG1b determines the ‘peak potential’ in mammalian cells.

A, Representative recordings of hERG1a, hERG1b and hERG1a/b channels expressed in HEK293 cells are shown. Currents were recorded using the protocol depicted in the inset. Only currents recorded during the ramp are shown. For comparison, currents were normalized to the maximum peak current during the ramp and plotted against the command voltage. The dotted lines indicate the potential where the currents peaked (‘peak potential’) and are also used to indicate the expressed channels. B, Summary data of the variation in ‘peak potential’. For comparison, the peak amplitude was normalized to an estimate of the number of channels (N) and plotted as a function of the ‘peak potential’.

Figure 3. The ‘peak potential’ is directly dependent on the relative abundance of hERG1 isoforms.

A, Representative recordings from X. laevis oocytes injected with different ratios of hERG1 isoforms. The same protocol as shown in figure 2A was used. Only currents recorded during the ramp are shown. For comparison, currents were normalized to the maximum peak current during the ramp and plotted against the command voltage. The dotted lines indicate the potential where the currents peaked (‘peak potential’) and are also used to indicate the isoform ratio (in % of hERG1b abundance). B, Summary data of the variation in ‘peak potential’. For comparison, the peak amplitude was normalized to N and plotted as a function of the ‘peak potential’.

Figure 4. The experimental data is reproduced by a hERG1 Markov model.

A, The effect of changes in transition rates, representing a gradual increase in the relative abundance of hERG1b, on simulated hERG1 currents. The transition rates of hERG1 channel activation (α₃), deactivation (β₃) and recovery from inactivation (β₄) were modified to represent a gradual increase in the relative abundance of hERG1b (see table S2 for details). The model was subjected to the same voltage protocol as in figure 2A. Only currents recorded during the ramp are shown. For comparison, currents were normalized to the maximum peak current during the ramp and plotted against the command voltage. The dotted lines indicate the potential where the currents peaked (‘peak potential’) and are also used to indicate the isoform ratio (in % of hERG1b abundance). B, The variation in ‘peak potential’ amplitude is shown as a function of ‘peak potential’. The data were not normalized as the number of channels in the model remained constant.

Figure 5. Action potential duration is modulated by the relative abundance of hERG1a and hERG1b.

A, Steady-state action potentials simulated by the modified ten Tusscher (TTF) model paced at a basic cycle length (BCL) of 1000 ms. The transition rates of hERG1 channel activation (α₃), deactivation (β₃) and recovery from inactivation (β₄) were modified to represent a gradual increase in the relative abundance of hERG1b (see table S2 for details). B, Simulated I _Kr corresponding to the action potentials shown in A. Legend is the same as in A. C and D, Channel state occupancy during the action potential is shown for the open and inactivated states respectively. Legend is the same as in A.

Figure 6. Restitution slope is modulated by the relative abundance of hERG1a and hERG1b.

A, Single-cell action potential restitution. The restitution curves were obtained using an S1-S2 restitution protocol measured at a BCL of 1000 ms. The action potential duration (APD₉₀) is plotted as a function of the diastolic interval (DI). The transition rates of hERG1 channel activation (α₃), deactivation (β₃) and recovery from inactivation (β₄) were modified to represent a gradual increase in the relative abundance of hERG1b (see table S2 for details). B, The slopes of the restitution curves were calculated by differentiation of single-exponential functions fitted to the curves in A and plotted as a function of DI.